This invention relates to the Magnetic Resonance Imaging (M.R.I.), a technique used in the medical diagnosis field for a number of years, to rapidly detect a series of anomalies and/or pathological conditions of living human or animal body organs or tissues. (i. e.: Stark D. D., Bradley W. G. Jr., Eds.: xe2x80x9cMagnetic Resonance Imagingxe2x80x9d, the C.V. Mosby Company, St. Louis, Mo. (USA), 1988). In particular, the invention relates to new chelating agents, especially aminopolycarboxylic acid derivative compounds and to metal chelates thereof with bivalent or trivalent paramagnetic ions and/or salts thereof as well as their use as M.R.I. contrast agents.
Diagnostic imaging techniques, such as Magnetic Resonance Imaging have been used in medical diagnosis for a long time. The use of contrast media to improve tissue differentiation, to delineate structures or monitor physiological functions constitutes in some cases a fundamental contribution in the best formulation of some medical diagnosis and a valid support for radiologist work.
The medical use of aminopolycarboxylic acid or carboxylic acid derivatives and metal chelates thereof as M.R.I. contrast agents is well known. Said contrast agents, to simplify, can be seen as pertaining to two main groups: the linear and the cyclic ones.
The present invention relates to linear polyaminopolycarboxylic acid derivatives, as well as their complexes with paramagnetic metal ions, in particular the Gd3+ ion.
Patent literature is rich in patent and patent applications relating to the use of linear polyaminopolycarboxylic acid derivatives in the preparation of MRI contrast agents. These compounds generally are derived from the simplest one, N,N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x3-diethylenetriamine-pentaacetic acid, (DTPA), of which the Meglumine salt of the Gd3+ complex has been commercialised for a number of years as MAGNEVIST(copyright). To improve stability, water solubility and selectivity and to reduce toxicity of these contrast agents generally patent literature proposes the preparation of esters or amido derivatives of said acids or the introduction of substituents on the diethylene unit of the diethylenetriamine DTPA skeleton. As an example of said patent literature we can cite: Guerbet EP 661279; Concat Ltd., WO 95/05118; Dibra WO 95/15319; Mallinckrodt WO 94/08630; Green Gross Corp. JP 06016606 and JP 05229998; Mallinckrodt U.S. Pat. No. 5,141,740 and U.S. Pat. No. 5,077,037; Cockbain-Nycomed WO 91/15467 and WO 92/11232; Salutar U.S. Pat. Nos. 4,889,931 and 4,858,451; Abbot Laboratoires EP 279307; Nycomed EP 299795; Metasyn Inc. WO 95/28179; Schering EP 680 464; and document cited in these patent publications. Some documents further exist in which substituents have been introduced in à to one or more carboxylic DTPA groups; for example: Bracco EP-B-230893 and U.S. Pat. No. 5,182,370; Schering WO 96/16928, WO 96/16929, WO 96/26180 and DE 4341724 enclosing à derivatives, generally comprising an aromatic group, particularly useful for the imaging of the hepatobiliary system. In particular, some patent literature further exist, in which the introduction of an aromatic or lipophilic group on the chelant structure is specifically stated to make the contrast agent particularly useful for a best definition of the liver and the biliary duct: the General Hospital Corporation U.S. Pat. No. 4,899,755 and WO-A-86/06605.
Among the other relevant documents disclosing large substituents on a DTPA like skeleton, in particular on the at position to the carboxy groups of DTPA, the following can also be mentioned. U.S. Pat. No. 5,746,995 describes chelates compounds in which the DTPA skeleton is substituted on the xcex1 position of one of the carboxy residues of the terminal/lateral part of the molecule. Said substituents are lipophilic radicals that render the claimed compounds particularly useful for the imaging of liver, gallbladder and bile ducts. U.S. Pat. No. 5,672,335 discloses substituted DTPA derivatives, which carry substituents that render the same sufficiently lipophilic to be useful for the imaging of liver and the biliary tracts. These compounds are specific hepatobiliary contrast agents for X-ray imaging (computer tomography). U.S. Pat. No. 5,514,810 discloses a process for preparing complexses substituted in the xcex1 position of the central carboxylic acid of the molecule. Said compounds are in any case lipophilic tetraesters of the four terminal carboxylic acids of the skeleton of DTPA. CA 2,177,977 discloses a number of complex derivatives of DTPA which carry huge substituents containing one or more aromatic moieties, which are especially suitable for diagnostic radiology of the liver.
No mention is found in this prior-art on the possibility that said compounds form strong non-covalent bonds with the serum/plasma proteins, turning out to be useful also as blood pool contrast agents. In fact the fact that said compounds are substantially hepatospecific means that they are preferentially, if not selectively taken up by the liver hepatocytes and are then excreted via the biliary route, thus being cleared away from the vessels in a very short time.
The compounds of the present invention are diethylenetriaminepentaacetic acid derivatives characterised by having a hindering group in a to at least one of the 5 DTPA carboxylic groups wherein said substituent has the dimension of a C1-C20 alkyl, linear or branched, saturated or unsaturated chain, which is substituted or interrupted by at least two cyclic, optionally aromatic, carbocyclic or eterocyclic, saturated or unsaturated, isolated or fused units.
Particularly preferred compounds are those having one hindering group in the xcex1 position of the central carboxylic acid of the DTPA skeleton, in particular the ones in which said hindering group is a tyrosine residue.
Said hindering group is probably responsible for the interaction of the paramagnetic chelates with biological components of the fluids in which the agent diffuses, wherein said interaction produces the surprisingly high relaxivity values that we have measured in Human Reconstructed Serum.
Relaxivity values of the contrast agent of the present invention have been tested either in saline or in human serum obtained by Seronorm(trademark) Human, freeze-dried human serum produced by Nycomed Pharma AS, Oslo, Norway. Serum obtained from said Seronorm(trademark) is substantially equivalent to the fresh one, so its use in the relaxivity determination grants a good picture of the xe2x80x9cin vivoxe2x80x9d behaviour and, further, an excellent reproducibility of this test.
The compounds object of the present invention are characterised by very high r1 and r2 relaxivity values. When measured in Seronorm(trademark) Human at 20 MHz, at a temperature of 39xc2x0 C., and at a concentration comprised from 0 to 1 mM, compounds of the present invention usually have r1 relaxivity equal to or, preferably, higher than 15sxe2x88x921mMxe2x88x921, thus confirming the formation of unexpectedly strong non-covalent bonds with serum proteins. This feature has been further confirmed by measuring the protein binding of the chelate complexes of the invention to Human Serum Albumin (HSA), whereas an average percent binding value superior to 80% was found. Last, significative amounts of the preferred compounds of the invention have also been found in the urines, showing that, thanks to their binding to HSA, the same are very promising as blood pool diagnostic agents.
The present invention relates to novel chelating agents, more particularly linear aminopolycarboxylic acid derivatives chelants, and metal chelates thereof and the use of such chelating agents and chelates in the preparation of diagnostic imaging contrast agents and in particular of contrast agents exhibiting improved serum relaxivity.
Said compounds are polyaminopolycarboxylic acid derivatives of formula (I) 
in which:
R is H, or a linear or branched, saturated or unsaturated C1-C20 alkyl, optionally interrupted by one or more xe2x80x94CH(OH)xe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94CH(NH2)xe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, SO2NHxe2x80x94 groups and/or one or more N, O, S atoms, optionally substituted with one or more xe2x80x94COOH groups and/or amide or ester derivatives thereof, and in which said alkyl chain is interrupted or substituted by at least 2, which are independently the same or different, isolated or fused, cyclic L residues, with the proviso that, when some L residues are fused together, the resulting polycyclic unit comprises no more than 3 cyclic group, and in which
L is a carbocyclic or heterocyclic, saturated or unsaturated or aromatic cyclic unit, comprising from 5 to 6 atoms, optionally substituted by one or more X groups, which are independently the same or different, in which
X is OH, halogen, NH2, NHZ, N(Z)2, xe2x80x94OZxe2x80x94, xe2x80x94SZ, xe2x80x94COZ, where the Z groups can independently be a C1-C5 linear or branched alkyl, optionally substituted with one or more xe2x80x94OH, xe2x80x94COOH or alkoxy groups,
or said X group is a xe2x80x94COOH group or a derivative thereof, such as an ester or an amido group, or an xe2x80x94SOZH group or an amido derivative of the same;
R1 is the same as R with the provisos that:
R and R1 cannot be at the same time H;
when R is different from H, R1 is H;
when R1 is different from H, R is H.
The compounds comprised within formula (I) can be either racemic or optically active.
The invention further comprises complexes of the ligand of formula (I) with metal ions of atomic number from 20 to 31, 39, from 42 to 44, 49 and from 57 to 83; particularly preferred metals being: Fe(2+), Fe(3+), Cu(2+), Cr(3+), Gd(3+), Eu(3+), Dy(3+), La(3+), Yb(3+), Mn(2+); as well as, where the metal chelate carries an overall charge, a salts thereof with a physiologically acceptable counterion, preferably selected from organic bases such as a primary, secondary or tertiary amines, a basic amino acid, or an inorganic base derived from an alkali metal or alkaline-earth metal cation such as: Na+, K+, Mg2+, Ca2+ or a mixture thereof.
The present invention further relates to the use of the compounds of formula (I) and of the salts of the complexes thereof as well as the pharmaceutical formulations containing them for a diagnostic or therapeutic scope.
Particularly preferred is their use as contrast agents in a method of performing the diagnostic imaging of the blood pool compartment.
Preferred are the compounds of formula (I) in which R or R1 are selected from the following groups: 
Among the compounds of formula (I) particularly preferred are the ones of formula (II), 
in which R1 is H and R is as defined above in formula (I), but is different from H.
Among compounds of formula (II), preferred are the compounds of formula (III): 
wherein:
Rxe2x80x2=independently H, halogen;
Rxe2x80x21=H, OH, N(Rxe2x80x3)2, COORxe2x80x3, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H, xe2x80x94SO2NHRxe2x80x3, C1-C6 alkyl, C1-C6 alkoxy;
A=direct bond (i.e. non intervening atom), xe2x80x94Oxe2x80x94, Cxe2x95x90O
m=integer 1-6;
n=integer 0-2;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups;
with the proviso that, when Rxe2x80x21=H, at least one of the substituents Rxe2x80x2 is different from hydrogen.
Among compounds of formula (III), particularly preferred are the compounds of formula (IV) 
where:
Rxe2x80x2=independently H, halogen;
Rxe2x80x21=H, OH, N(Rxe2x80x3)2, COORxe2x80x3, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H, xe2x80x94SO2NHRxe2x80x3, C1-C6 alkyl, C1-C6 alkoxy;
m=integer 1-6;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups;
with the proviso that at least one of the substituents Rxe2x80x2 is different from hydrogen,
as well as compounds of formula (V) 
xe2x80x83where:
Rxe2x80x21=OH, N(Rxe2x80x3)2, COORxe2x80x3, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H, xe2x80x94SO2NHRxe2x80x3, C1-C6 alkyl, C1-C6 alkoxy;
m=integer 1-6;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups.
Among compounds of formula (II), preferred are also those of formula (VI) 
where:
R2=C1-C8 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, S atoms, optionally substituted with xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2 groups, said alkyl being interrupted or substituted with a polycyclic unit comprising from 2 to 3 saturated or unsaturated or aromatic fused rings, said polycyclic unit being interrupted by one or more N, O, S and optionally substituted with xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, C1-C6 alkyl, C1-C6 alkoxy, C6-C20 arylalkoxy groups;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups;
and particularly preferred are the compounds of general formula (VII) 
xe2x80x83in which:
R3=a polycyclic unit comprising from 2 to 3 saturated or unsaturated or aromatic fused rings, said polycyclic unit being interrupted by one or more N, O, S and optionally substituted with xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, C1-C6 alkyl, C1-C6 alkoxy, C6-C20 arylalkoxy groups;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups;
n=integer 1-6.
Two further groups of preferred compounds, comprised within formula (II), are the compounds of formula (VIII) 
in which:
m=integer from 1 to 4;
n=independently integer from 0 to 2;
R4=independently saturated, unsaturated or aromatic ring, optionally interrupted by one or more N, O, S atoms and optionally substituted with one or more xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups;
and the compounds of formula (IX) 
xe2x80x83in which:
R5=C1-C3 alkyl, interrupted or substituted with 2 to 3 saturated, unsaturated or aromatic, isolated or fused rings, that are optionally interrupted by one or more N, O, S and optionally substituted with one or more xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups;
m=1-6.
Among compounds of general formula (IX), particularly preferred are the compounds of formula (X) 
in which:
R6=saturated, unsaturated or aromatic 5- or 6-membered ring, optionally interrupted by one or more N, O, S;
m=1-6;
n=2 or 3;
p=0 or 1;
with the proviso that p+n=3.
Among the compounds of formulae (III) and (IV), most preferred are the compounds from 1 to 3 of formula: 
Among the compounds of formula (V), most preferred is compound 4 of formula: 
Among the compounds of formula (VI), most preferred is compound 5 of formula: 
Among the compounds of formula (VII), most preferred is compound 6 of formula: 
Among the compounds of formula (VIII), most preferred are compounds 7 and 8, respectively of formula: 
and among the compounds of formulae (IX) and (X), most preferred are compounds from 9 to 11 of formulae 
respectively.
The preparation of the compounds of the present application comprises the regiospecific introduction of the hindering substituent in a to a carboxylic group of the acetic acid bound to the central nitrogen atom of DTPA.
One of the preferred synthetical ways used refers to that introduced by Rapoport (J. Org. Chem. 1993, 58, 1151-1158), starting from natural or synthetical à-amino acid derivatives. An alternative way comprises the use of synthons such as glutamic acid or lysine, which allows the introduction of hindering groups quite distant from the carbon atom in à to a carboxylic group of the central acetic acid residue, exploiting the terminal acid or amino functions, respectively, of a.m. amino acids.
Starting from suitable precursor synthons it is also possible make use of the synthesis disclosed in U.S. Pat. No. 5,514,510.
As far as the introduction of the hindering substituent at the à- position to the carboxylic group of one of the acetic groups bound to the side nitrogen atoms of DTPA is concerned, the synthesis scheme below can be followed: 
wherein R1 is as defined above for compounds of general formula (I).
The synthesis comprises the following steps:
(a) precursor (1), wherein X=Cl, Br or other leaving groups, is reacted with a diethylenetriamine excess in water, at a temperature of about 50xc2x0 C., to obtain almost selectively compound (2), which is reacted in step
(b) with sodium bromoacetate in water at pH 10, to give the pentaacid (3), which is reacted, in the subsequent step
(c) with a suitable oxide or salt of a metal having atomic number comprised from 20 to 31, 39, from 42 to 44, 49 and from 57 to 83 (such as Gd2O3, GdCl3) e with the appropriate amount of a physiologically acceptable organic base (such as meglumine) or of an inorganic base the cations of which are sodium, potassium, magnesium, calcium, or mixtures thereof, to give the final compound (4),
xe2x80x83wherein:
Men+=ion of the metal element having atomic number comprised from 20 to 31, 39, from 42 to 44, 49 and from 57 to 83 (such as Gd3+);
n=number of the positive charges of said ion;
m=number of the overall negative charges of the metal chelate;
Bz+=Na+, K+, Mg++, Ca++ or mixtures thereof, or it is the salt of a physiologically acceptable organic base;
z=number of the positive charges of B;
p=an integer so that: pxc3x97z=m.
Table I above discloses the high relaxivity shown in serum by the compounds of the present application; r1 and r2 relaxivity values of some of the preferred compounds are reported, in comparison with the corresponding r1 and r2 values measured for some of the mayor prior-art compounds: Gd-DTPA Dimeglumine salt (MAGNEVIST(copyright)); Gd-BOPTA Dimeglumine salt and Gd-EOB-DTPA Dimeglumine salt.
The data of Table 1 clearly show that the compounds of the present invention have surprisingly high relaxivity values r1 and r2, measured in Seronorm(trademark) Human.
The same experiments were performed on the chelate complex compounds described in Examples 11 to 17 (Compounds 12 to 18). Values of r1 and r2, measured in Seronorm(trademark) Human are superior to 31 and 35, respectively. The percentual binding to HSA is on average superior to 80% up to 97%. The urinary elimination in rats is even superior to 38%, for some of the preferred compounds.
This is particularly interesting from the application point of view, both as far as the improvement in the obtainable images, the development of formulations specific to particular districts, i.e. the blood pool district, and the determination of optimum low dosages of the contrast medium are concerned.